This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The study of protein phosphorylation by mass spectrometry often requires enrichment of low abundance proteins and peptides from complex biological samples. One method of enrichment is immobilized metal-ion affinity chromatography (IMAC). Non-specific binding is a complication in IMAC enrichment, and can be reduced by treatment with methanolic HCl to esterify peptide acidic groups. The side reactions that occur during esterification have been investigated to document the conditions that lead to their occurrence and minimize their impact. The use of alternative metal oxides has been explored, as well as precipitation of phosphopeptides/ proteins with calcium salts. Each procedure has been optimized by use of standards and then applied to the analysis of samples from biological sources. Procedures have been developed for efficient detection of phosphopeptides during LC/MS analysis on the LTQ-Orbitrap tandem MS. A new dissociation method, electron transfer dissociation (ETD) is being used on the Bruker ion trap and has provided excellent, site-specific results for standards and previously characterized peptides.With support from a supplement to the P41 grant, it is now being added to the Orbitrap system. This system is being used for the analysis of phosphopeptides for which at least some of the sites are as yet unknown.